Published: June 10th, 2013
We present a user-friendly, high-throughput operant system for the evaluation of pain behaviors in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) can assess pain through a reward/conflict paradigm thus providing a more humane way of testing. This protocol will yield more clinically relevant and translational data from rodents.
We present an operant system for the detection of pain in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) assesses pain behaviors in a more clinically relevant way by not relying on reflex-based measures of nociception. Food fasted, hairless (or shaved) rodents are placed into a Plexiglas chamber which has two Peltier-based thermodes that can be programmed to any temperature between 7 °C and 60 °C. The rodent is trained to make contact with these in order to access a reward bottle. During a session, a number of behavioral pain outcomes are automatically recorded and saved. These measures include the number of reward bottle activations (licks) and facial contact stimuli (face contacts), but custom measures like the lick/face ratio (total number of licks per session/total number of contacts) can also be created. The stimulus temperature can be set to a single temperature or multiple temperatures within a session. The OPAD is a high-throughput, easy to use operant assay which will lead to better translation of pain research in the future as it includes cortical input instead of relying on spinal reflex-based nociceptive assays.
Chronic, uncontrolled pain remains a major public health problem and novel analgesic treatments often fail to translate from the bench to bedside. This lack of success is partly due to the use of inefficient behavioral assays in reflex based measures of pain which do not necessarily or completely model the human pain condition 1,2, and specifically, a lack of a reliable, high-throughput, commercially available, in vivo pain-assessment assay for both rats and mice. We present here a high throughput, easy to use version of our operant based nociception assay. This new system is based on our previous operant orofacial pain assay which has been demonstrated to be sensitive to detecting different pain modalities including heat, cold, and mechanical 3,4,5. From these measures, a wide variety of fields have been studied, including analgesics 6,3,4,5,7, pain conditions like inflammation, hyperalgesia, and allodynia 3,8, trigeminal neuralgia 9, and peripheral nociceptive modulation via TRP channels with capsaicin, resiniferatoxin, menthol, and icillin 8,10,5,11. Psychological effects like anxiety-induced modulation of pain 12 and the placebo effect 13have also been demonstrated with orofacial operant testing suggesting it may be appropriate for measuring the full experience of pain and not simply nociception.
The Orofacial Pain Assessment Device (OPAD) uses a reward/conflict assay which allows a rodent to choose between receiving a reinforcing reward or escaping an aversive stimulus thus controlling the amount of pain it feels during a session 14,15. Rodents are first trained to press their faces into temperature controlled thermodes in order to gain access to a food bottle containing a liquid reward. After training, the stimulus temperature can be heated or cooled and differences in responding can indicate the level of nociception or analgesia the animal perceives. The OPAD is also capable of rapid changes in temperature which allows baseline testing and the assessment of pain at hot and cold temperatures within a single testing session. Here we present a simple protocol which highlights the OPAD's ability to detect changes in pain caused by heat, cold, and the TRPV1 agonist capsaicin 16. Capsaicin is used below as a thermal sensitizing agent because it has several benefits to this assay as it is non-tissue damaging and has been demonstrated previously to induce facial allodynia and hyperalgesia in rodent models 8. We will demonstrate how the OPAD software can rapidly obtain, analyze, graph, and perform statistical analyses on rodent behavioral data.
Here the use of the OPAD (Stoelting Co., Wood Dale, IL) is described in general terms for an example experiment using capsaicin. The operator has the freedom to program numerous experiments with many options and pain models though. For instance, the administration of analgesics reduce nociceptive measures 6,3,4,5,7 and other pain models like chronic constriction injury produce and inflammation increased nociceptive behaviors 3,9. These models are easily adaptable to the following protocol.
For all experiments, maleSprague-Dawley rats (250-300 g, Charles River, Raleigh, NC) were used. These were housed in pairs in 22 °C temperature and 31% humidity controlled rooms with a normal 12-hr light/dark cycles (6am-6pm lights on) and had free access to food and water except when fasted. Behavioral sessions were performed during the light phase. These facilities were AAALAC accredited and all procedures were approved by the University of Florida IACUC.
1.Training and Baseline Sessions
2. Pretest Preparation and Capsaicin Treatment
3. Programming the OPAD System for Protocols and Experiments
4. Running the Assay
5. Analyzing, Graphing, and Statistical Analysis of Data with OPAD Software
6. Clean Up
Typical results are illustrated for a single rodent's behavior on the OPAD in Figures 1A-D. The number of licks is high for every segment of the session at the neutral 33 °C temperature, but low for aversive ones (45 °C and 7 °C) as illustrated in Figure 1A. As Figure 1B demonstrates, long bouts of contact are made at 33 °C as is typical for non-nociceptive stimulus temperatures. The duration decreases and the number of contacts increase during periods where the temperatures are painful. Figure 1C is a diagram of the ramping protocol the OPAD was programmed to use for all test sessions. Figure 1D displays the total amount of reward ingested over time in grams. Similarly to the number of licks, animals prefer the neutral temperatures over the painful ones. The lick/face ratio (L/F) for the baseline session was calculated by the OPAD and is illustrated in Figure 1E. This ratio is much higher during the three non-painful 33 °C sessions (20-46 licks per facial contact) than at the painful sessions of 45 °C (3 licks per facial contact) and 7 °C (1 lick per facial contact). A Repeated Measures One-Way ANOVA was significant (F(4,52)=6.2182, p<0.001) for an effect of temperature on the L/F ratio. Bonferroni's test were significant when comparing 33 °C vs. 7 °C (p<0.05), 45 °C vs. 33 °C (2) (p<0.01), and 33 °C (2) vs. 7 °C (p<0.01). N=16 for all temperatures. In Figure 1F the capsaicin treated rodents (N=8) were not significantly different from naive rats (N=8) at any of the neutral 33 °C temperatures. Capsaicin treated rodents did have a significantly lower L/F ratio at 45 °C (t-test, t(13)=2.9350, p=0.012). The capsaicin group had higher L/F ratios at 7 °C, but this was not significant.
Figure 1. Measuring nociception with the OPAD. A single rodent's behavior on the OPAD is graphed for A) number of licks, B) contacts, C) temperature of the thermode during the session, and D) reward intake in grams. E) The lick/face ratio is high during the three non-painful 33 °C sessions and is significantly lower at the painful sessions of 45 °C and 7 °C (Repeated Measures One-Way ANOVA, F(4,52)=6.2182, p<0.001, Bonferroni's test 33 °C vs. 7 °C (p<0.05, #), 45 °C vs. 33 °C (2) (p<0.01, **), and 33 °C (2) vs. 7 °C (p<0.01, ##). F) Capsaicin treated rodents had a significantly lower L/F ratio at 45 °C (t-test, t(13)=2.9350, p=0.012, *) but at none of the neutral temperatures. N=16 for 1E and N=8 for capsaicin and N=8 for naive for 1F. Click here to view larger figure.
The OPAD system is an easy to use, high throughput assay capable of detecting changes in pain perception in rodents. The high throughput nature of this system means that numerous animals can be tested in a single day by a single person. This is due to the OPAD software system as it allows up to 16 boxes to be run concurrently on a single computer. This means that after the initial setup time, about 48 operant runs (at 18 min per run) can be performed an hour, even more if the session time is set to less time per stage. This allows for pain testing in hundreds of animals a day. This amount of testing would not be practical with most traditional pain assays.
Consistent with our previous work, rodent behavior is altered under painful conditions. During the non-noxious periods rodents typically have long bouts of drinking in which they maintain contact with the thermodes. During aversive 45 °C or 7 °C conditions, the rodents have much shorter bouts as they cannot maintain contact for long periods of time. Therefore the lick/face ratio (number of licks divided by the number of facial contacts within a session) alters with pain. Capsaicin increased the sensitivity to heat pain as demonstrated by a lower L/F ratio in the treated versus untreated rodents at the 45 °C temperature. Analgesics can return this lick/face ratio to levels similar to non-painful conditions 3. Although pain conditions that are easily produced on skin (like the application of capsaicin cream) are the simplest methods of detecting pain on this assay, animal models of more clinically relevant deep neural tissue pain like trigeminal neuralgia can also alter behavior on operant orofacial assays 9. Taken together these data are supporting evidence that the OPAD is sensitive to alterations in heat and cold pain, pain thresholds, and noxious chemical agents like capsaicin in addition to the operant orofacial pain assay's ability to detect numerous other conditions of pain and analgesia 6,3,8,17,10,5,12,18,11,9.
The OPAD's system of measuring pain is a more clinically relevant, meaningful, and humane method of detecting pain than reflex-based measures. These traditional measures of nociception like the paw withdrawal with von Frey filaments 19 and the tail-flick assay 20 have been used for over a century but they only measure the response to an experimenter-inflicted stimulus. The animal has little control and the "nociception" is mainly localized to the spinal cord. For humans, the subjective pain experience is also important as people are simply asked to report their subjective levels of pain. The ability for animals to self-report their pain in operant based procedures would be a breakthrough for basic pain research 1. With the OPAD, animals are given the choice of whether to respond during a painful stimulus or not. If it is too painful, animals simply reduce their attempts to reach the reward and thus limit their exposure to pain. This is a much more humane and less stressful assay when compared to many reflex-based measures in which animals often have their movement restricted and have no control over the amount of painful stimuli to which they are exposed. The need to escape from pain is an inherent drive in all animals and the OPAD incorporates this behavior instead of compensating for it like other nociceptive assays. The movement away from reflex-based measures of pain into operant tasks is becoming more common in the field. Other groups have used non-reflex-based measures like examining meal duration 21,22,23 and thermal heat pain escape paradigms 24 (For a review of other pain measures we suggest our first reference 1). The OPAD is able to combine elements of these into a unified measure, the Lick/Face ratio, which examines food intake and the need to escape from painful stimuli. Another benefit is that this assay is capable of measuring pain over long periods of time (1-2 months) without losing sensitivity 7,9. Due to its advantages over reflex-based testing, this less stressful and more humane assay is well adapted to measuring long-term changes in nociceptive behavior in rodents.
Operant pain measures often give different results when compared to reflex-based measures in terms of opioid dose effects and pain thresholds. While high doses of opioids are typically used for reflex-based measures 25 several studies indicate that lower doses are needed for responses on operant assays 26,27,28. High drug doses could also interfere with operant measures but these are detectable with the OPAD 6. Other studies have also demonstrated that the thresholds for escape from a painful stimulus are different for operant versus reflex-based measures 29,2,30 suggesting a major difference between an animals' perception of pain versus the speed of their spinal reflexes. A benefit of the OPAD is that the rodent can choose whether or not to perform the task, this allows the rodent to express escape or avoidance behavior. This complex behavior requires cortical decision making to control the amount of nociception the rodent feels 14,29,15,30. While escape and avoidance behaviors can interfere with reflex based measures these pain behaviors are an integral component of the OPAD. The differences in pain thresholds and the lower doses of opioids needed for operant assays suggest a higher sensitivity to pain and analgesia than traditional reflex-based measures.
Although the OPAD can measure pain more directly than traditional assays, several experimental conditions and drugs could have an adverse effect on this assay and must be controlled for. Alterations in appetitive motivation can alter behavior on this assay. This could be reflected by a difference in the reward itself 31 or by the motivation for the reward 6. Care must be taken to ensure that the animal's motivation for the reward is constant as many drugs can interfere with motivation. For instance, high doses of morphine and other opioids can cause hyperphagia for sweet, fatty substances 32 which will alter responding on the operant orofacial assay 6. While this does suggest that this operant-based reward-conflict paradigm has wider implications for use including the fields of anxiety and addiction (i.e. changing the rewarding aspects in the presence of a given painful stimulus) it is important to control for appetitive alterations during pain testing sessions. These alterations in motivation do not appear at these clinically relevant lower doses but the analgesic effects remain intact 3. One way to control for this possible confound is to ensure the dose of the drug given does not increase behavior at a neutral (33-37 °C) temperature. Testing a drug versus a non-drug group at a neutral temperature should be a first step before adding a pain component. Also, given that several baseline sessions are possible within a test session using the OPAD these issues can be detected and can be controlled for within a single behavioral session. As the fasting schedule can alter motivation on this assay it is important to keep this consistent. We typically do an overnight fast, but other schedules are possible. For instance, we have experimented with a daily short fast of 6 hr before (unpublished results). This allows for testing daily instead of every other day. Also, unfasted rats have also responded well on the assay 9. Whatever fasting techniques are used it is mainly important to keep it consistent throughout testing to control for motivational factors.
In conclusion, the OPAD is an easy to use operant assay which measures pain on a level much more similar to the human condition than traditional pain assays. The key feature of this system is integration of the experimental parameters and protocols, data acquisition, and analysis/outcome measures using a software-controlled system. This will provide a wealth of user-controlled options and parameters to collect and analyze numerous outcome measures in a high-throughput fashion. This contrasts the commonly used pain-testing systems (e.g. tail flick, von Frey filaments) which are neither software-driven nor high-throughput. The software-driven system provides a significant advancement for how behavioral studies are designed and the how the data is collected and analyzed and an increase in the use of this assay will allow basic pain research to become more clinically translatable in the future. This system is expected to have a significant impact on advancing future research related to pain because these operant behavioral studies can provide the necessary link for understanding the influence of higher order structures on overall pain behavior.
Richard Mills and Chris Lloyd are employees of Stoelting Co.
Production and Free Access to this article is sponsored by Stoelting Co.
National Institute on Drug Abuse, NIH grant 5R44DA026220-03
|Name of Reagent/Material
|Sweetended Condensed Milk
|Opthalamic vet ointment
|Hair remover lotion
|Church and Dwight Co., Inc
|Additional OPAD cages
|Cotton tipped applicators
|Fluotec 4 Vaporizer
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